Method for producing recombinant of methanol-assimilating bacterium

ABSTRACT

A recombinant of a methanol-assimilating bacterium in which an exogenous linear DNA fragment is introduced into its chromosomal DNA, and is prepared by the following steps:  
     (a) preparing an exogenous linear DNA fragment comprising a nucleotide sequence identical to a nucleotide sequence of an arbitrary region of said chromosomal DNA,  
     (b) introducing the linear DNA fragment into the methanol-assimilating bacterium to obtain recombinants, and  
     (c) selecting a recombinant in which said region on the chromosome is replaced with said linear DNA fragment.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for producing a recombinant of a methanol-assimilating bacterium. More precisely, the present invention relates to a method for producing a recombinant whereby a gene on a chromosome is replaced with an exogenous gene. The present invention is useful in methods of breeding or improving methanol-assimilating bacteria.

[0003] 2. Description of the Related Art

[0004] In order to delete, amplify or modify a desired gene on a chromosome of a methanol-assimilating bacterium by gene substitution, typically a method of incorporating a recombinant plasmid which is capable of conjugative transfer and which carries a DNA segment containing the desired gene into a plasmid DNA donor bacterium is utilized. This method enables conjugative transfer of the recombinant plasmid to a methanol-assimilating bacterium. Alternatively, a method of introducing a plasmid DNA having a DNA segment containing a desired gene into a methanol-assimilating bacterium by electroporation and causing homologous recombination between the desired gene on a chromosome and the DNA segment on the introduced plasmid may be utilized. Examples thereof which have been disclosed to date include, for example, disruption of an desired gene in a Methylobacterium extorquens strain having the serine pathway or a Methylobacillus flagellatus strain having the ribulose monophosphate pathway (J. Bacteriol., vol. 176, pp.4052-4065 (1994), Microbiology, vol. 146, pp.233-238 (2000)).

[0005] Methods described above use circular DNAs and are useful in gene substitution for many procaryotes (Nature, vol. 289, pp.85-88 (1981)), however, it is believed that methods for substitution of a desired gene using linear DNAs, which will be described herein, are inapplicable to most procaryotes (Proc. Natl. Acad. Sci. USA, vol. 97, pp.6640-6645 (2000)).

[0006] The gene substitution technique using linear DNA has been exclusively used for yeast, fungi, Bacillus subtilis, and the like. This method is extremely simple and advantageous in that it does not require a series of time-consuming operations, as is required in methods using circular DNA, i.e., the first homologous recombination reaction of a circular DNA and a homologous region on a chromosome, second homologous recombination reaction and selection of a recombinant in which the desired gene as a target is replaced from a group of obtained recombinants. Furthermore, in many cases, the desired gene substitution does not occur in the recombinants obtained by the second homologous recombination reaction, and they return to the gene structure before the operations, which results in the pain-staking selection of a strain having the desired gene structure occurring at a low frequency from many recombinants.

[0007] However, in Escherichia coli, the methods for substitution of a desired gene using a circular DNA have constituted the mainstream methods. It is said that this is because Escherichia coli has a powerful enzymatic activity for degrading introduced linear DNA. Therefore, in Escherichia coli, gene substitution, deletion and modification using a linear DNA have been possible only in strains in which the enzymatic activity is reduced (Marinus M.G., et al., Mol. Gen. Genet., 192, pp 288-289 (1983), Russell C. B., et al., J. Bacteriol., 171, pp.2609-2613 (1989)).

[0008] Furthermore, in methanol-assimilating bacteria, only gene substitution methods using a circular DNA have been known to date.

SUMMARY OF THE INVENTION

[0009] An object of the present invention is to provide a simple gene substitution method for breeding and improvement of methanol-assimilating bacteria.

[0010] It is a further object of the present invention to provide a method for producing a recombinant of a methanol-assimilating bacterium in which a exogenous linear DNA fragment is introduced into the chromosomal DNA of the methanol-assimilating bacterium comprising:

[0011] (a) preparing an exogenous linear DNA fragment comprising a nucleotide sequence identical to a nucleotide sequence of an arbitrary region of said chromosomal DNA,

[0012] (b) introducing said linear DNA fragment into the methanol-assimilating bacterium to obtain recombinants, and

[0013] (c) selecting a recombinant in which said region on the chromosome is replaced with said linear DNA fragment.

[0014] It is a further object of the present invention to provide a method as described above, wherein said methanol-assimilating bacterium is a Methylophilus bacterium.

[0015] It is a further object of the present invention to provide a method as described above wherein said methanol-assimilating bacterium is Methylophilus methylotrophus.

[0016] It is a further object of the present invention to provide a method as described above, wherein said linear DNA fragment comprises a segment having said nucleotide sequence identical to the arbitrary region of said chromosomal DNA, and another sequence inserted into the segment.

[0017] It is a further object of the present invention to provide a method as described above, wherein said linear DNA fragment comprises partial deletion or substitution of one or more nucleotides.

[0018] According to the present invention, transformation, especially gene substitution, of methanol-assimilating bacteria can be efficiently performed in a simple manner.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The inventors of the present invention considered that if gene substitution utilizing an exogenous linear DNA can be carried out in methanol-assimilating bacteria, genetic manipulation, including chromosomal manipulation, should be possible in the breeding of methanol-assimilating bacteria for industrial use, and thereby time and cost are significantly saved. Thus, the inventors assiduously studied in order to achieve the aforementioned objects. As a result, they found that, in Methylophilus bacteria, a recombination reaction efficiently occurred between DNAs having the same sequences even when they were linear DNAs, and therefore substitution, deletion and modification of a desired gene could be possible. Thus, they accomplished the present invention.

[0020] Hereafter, the present invention will be explained in detail.

[0021] The present invention provides a method for producing a recombinant of a methanol-assimilating bacterium. The present invention also provides a method for transformation of a methanol-assimilating bacterium, or a method for gene substitution in a methanol-assimilating bacterium.

[0022] The methanol-assimilating bacterium in the present invention is a bacterium capable of utilizing methanol as a carbon source, and a strict methanol-assimilating bacterium is preferred. Examples of strict methanol-assimilating bacterium include, for example, Methylophilus bacteria, which are gram-negative bacilli, such as Methylophilus methylotrophus, Methylobacillus bacteria such as Methylobacillus glycogenes and Methylobacillus flagellatum, and so forth. Specific examples include the Methylophilus methylotrophus AS1 strain (NCIMB 10515), Methylobacillus glycogenes NCIMB 11375 strain, ATCC 21276 strain, ATCC 21371 strain, ATR80 strain, A513 strain (described in Appl. Microbiol. Biotechnol., vol. 42, pp.67-72 (1994)), Methylobacillusflagellatum KT strain (described in Arch. Microbiol., vol. 149, pp.441-446 (1988)) and so forth. Among these, the NCIMB 10515 strain and NCIMB 11375 strain can be obtained from National Collections of Industrial and Marine Bacteria (Address: NCIMB Lts., Torry Research Station 135, Abbey Road, Aberdeen AB9 8DG, United Kingdom).

[0023] In the present invention, the “exogenous linear DNA fragment” means a DNA fragment which is to be introduced into the methanol-assimilating bacterium from the outside of the bacterium. The origin of the exogenous linear DNA fragment may be an organism other than the methanol-assimilating bacterium host, or may be the methanol-assimilating bacterium host itself, or a bacterium of the same species.

[0024] In the present invention, the “recombinant” means a methanol-assimilating bacterium in which the linear DNA fragment is introduced into its chromosomal DNA by homologous recombination. When the linear DNA fragment contains one or more nucleotide substitutions, the recombinant obtained by the method of the present invention may not be structurally distinguished from a mutant strain obtained by a mutagenesis treatment. However, such a recombinant is included in the “recombinant” as long as it is obtained by the method of the present invention.

[0025] The linear DNA used in the present invention is a linear DNA fragment comprising a nucleotide sequence identical to a nucleotide sequence of an arbitrary region of a chromosomal DNA (henceforth also referred to as a “target region”).

[0026] The “linear DNA fragment” means a DNA fragment having free 5′ end and 3′ end, and means that it is not a “circular DNA.” Furthermore, the actual form of the linear DNA fragment may not necessarily be linear, and it may have a bend or torsion. Although the linear DNA fragment may be double-stranded or single stranded when it is introduced into a Methylophilus bacterium, it is preferably double-stranded.

[0027] One embodiment of the linear DNA fragment used in the present invention includes, for example, a segment having a nucleotide sequence identical to that of an arbitrary region of a chromosomal DNA, as well as another sequence inserted into the segment. Examples of other sequences include the marker gene described later. Furthermore, in another embodiment, the linear DNA fragment has a nucleotide sequence identical to that of a target region, but includes a partial deletion or substitution of one or more nucleotides. In this embodiment, the portion other than the portion of the aforementioned deletion or nucleotide substitution of the linear DNA fragment preferably has the same nucleotide sequence as that of the target region.

[0028] The segment having the same nucleotide sequence as that of the target region can be obtained by cloning the target region on the chromosome of the methanol-assimilating bacterium. The target region may be, for example, cloned into a plasmid from the chromosomal DNA to obtain a recombinant plasmid, and then excised from the recombinant plasmid with a restriction enzyme, or it may be obtained by directly amplifying the desired fragment from the genomic DNA by PCR.

[0029] When such a linear DNA fragment as described above (henceforth also referred to as the “DNA fragment for introduction”) is introduced into a methanol-assimilating bacterium host, and a homologous recombination reaction occurs between a sequence on the host chromosomal DNA which is identical with at least a part of the DNA fragment for introduction (henceforth referred to as the “target region”) and the DNA fragment for introduction, insertion of the DNA fragment for introduction and removing of the target region occurs simultaneously. As a result, the target region is replaced by the DNA fragment for introduction.

[0030] When the aforementioned target region is a gene (henceforth referred to as a “target gene”), and the DNA fragment to be introduced is a gene having identity with the aforementioned gene but having a partially different sequence (henceforth referred to as a “gene for introduction”), the target gene is replaced with the gene for introduction (henceforth referred to as “gene substitution”). The gene for introduction may be a fusion gene consisting of two or more of genes or a gene complex containing two or more of genes.

[0031] When the aforementioned target region is a structural gene coding for a protein, and the gene for introduction does not code for any protein having an activity due to deletion of a partial sequence or insertion of another sequence (henceforth referred to a “disrupted-type gene”), the target gene is disrupted by the gene substitution. Furthermore, by modifying a nucleotide sequence of an expression regulatory sequence of the target gene, expression of the target gene can be reduced.

[0032] Furthermore, if a mutant gene having one or more arbitrary mutations is used as the gene for introduction, the arbitrary mutations can be introduced into the target gene by the gene substitution. This “introduction of mutations” includes replacement of mutations of the target gene contained in the methanol-assimilating bacterium with wild-type or other mutations.

[0033] The deletion, insertion or mutation in the aforementioned gene for introduction may concern either one nucleotide or a region consisting of two or more nucleotides. Such modifications of the gene for introduction can be performed by the site-specific substitution method.

[0034] In order to determine whether the substitution of the DNA fragment for introduction by the target region has occurred as intended, for example, a drug resistance marker gene having resistance to an antibiotic may be incorporated into the DNA fragment for introduction. However, such a marker gene needs to have sequences on either side that is identical to the target region. Drug resistance marker genes include, but are not limited to a gene imparting resistance to a drug such as kanamycin, gentamycin, tetracycline, ampicillin or streptomycin. Such a marker gene as described above can be used for construction of a disrupted-type gene by inserting it into the gene for introduction.

[0035] The disrupted-type gene inserted with a marker gene may be prepared by a gene recombination technique using a plasmid DNA as shown in the examples section, or it can be prepared by simultaneously performing amplification of the gene for introduction and insertion of the marker gene by crossover PCR.

[0036] When a strain in which the desired gene on a chromosome is replaced with the DNA fragment for introduction can be selected according to a phenotype or genotype, it is not necessarily required to use a drug resistance marker gene. A genotype may be easily confirmed by, for example, hybridization or PCR.

[0037] Furthermore, the length of the DNA segment identical to the target region in the DNA fragment for introduction needs to be of such a length that the homologous recombination can occur in the methanol-assimilating bacterium. Specifically, it is usually a length of 20 or more nucleotides, preferably 500 or more nucleotides, more preferably 1000 or more nucleotides. Such a DNA fragment can be recognized by an enzyme for homologous recombination in a host cell, and homologous recombination proceeds between the DNA fragment for introduction and the target region on a chromosome. The DNA fragment for introduction may include a DNA segment which is not identical to the target region in a region upstream and/or downstream from the DNA region identical to the target region.

[0038] When the gene for introduction contains a mutation or insertion, each of upstream and downstream regions of the mutation or insertion site preferably has a length of 500 or more nucleotides, and it is a length of around 5000 nucleotides at most. Furthermore, when a drug resistance marker gene is inserted into the gene for introduction, each of the segments identical to the target region at regions upstream and downstream from the marker gene preferably has a length of 500 or more nucleotides.

[0039] Furthermore, according to the gene substitution method of the present invention, it is also possible to introduce a gene that is not inherently contained in a methanol-assimilating bacterium host into an unnecessary gene on a chromosome. The DNA fragments having sequences identical to upstream and downstream regions of the unnecessary gene on a chromosome are ligated to the both ends of DNA containing the gene for introduction to prepare a linear DNA fragment, and the resulting linear DNA fragment is used to transform a methanol-assimilating bacterium. The DNA fragment introduced as described above causes a recombination reaction with genes on a chromosome identical to the nucleotide sequences of the both ends of the DNA fragment, thereby the unnecessary gene is deleted, and instead, a foreign gene is inserted at the site of the deletion.

[0040] As for the methods of digestion, ligation and hybridization of DNA, PCR and so forth, usual methods well known to those skilled in the art can be used. Such methods are described in Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press (1989) and so forth.

[0041] Examples of the method for introducing the exogenous linear DNA fragment into a methanol-assimilating bacterium include, but are not limited to electroporation (described in Canadian Journal of Microbiology, 43, 197 (1997)) and so forth. For example, the exogenous linear DNA fragment can be introduced into a methanol-assimilating bacterium using a commercially available apparatus (GenePulser produced by BioRad etc.) according to a method defined for the apparatus.

[0042] A strain in which substitution of a target gene on a chromosome has occurred can be selected based on a phenotype of the target gene, gene for introduction or marker gene, or a genotype of each gene.

[0043] For example, in the case of Methylophilus methylotrophus, the SEII medium described in the following examples can be used as a base medium used for evaluation of drug resistance. By adding an appropriate drug to the base medium and culturing the bacterium at an appropriate temperature in the range of 20 to 40° C. for about 12 to 100 hours in the medium to select a strain resistant to the drug, a strain in which a desired gene is replaced with an introduced DNA fragment can be obtained.

EXAMPLES

[0044] Hereinafter, the present invention is explained more specifically, with reference to the following non-limiting examples.

Example 1 Method for Disrupting RecA Gene Using Linear DNA

[0045] A recA gene-deficient strain was constructed from a wild-type strain of Methylophilus methylotrophus, the AS1 strain (NCIMB No. 10515), using a linear DNA as follows.

[0046] The AS1 strain was inoculated into 50 mL of the SEII medium (composition: 1.9 g/L of K₂HPO₄, 5.0 g/L of (NH₄)₂SO₄, 1.56 g/L of NaH₂PO₄.2H₂O, 0.2 g/L of MgSO₄.7H₂O, 0.72 mg/L of CaCl₂.6H₂O, 5 μg/L of CuSO₄.5H₂O, 25 μg/L of MnSO₄.5H₂O, 23 μg/L of ZnSO₄.7H₂O, 9.7 mg/L of FeCl₃.6H₂O, 1% (v/v) of CH₃OH) and cultured overnight at 37° C. Then, the culture broth was centrifuged to collect the cells. Chromosomal DNA was purified from the obtained cells using a commercially available kit (Genomic DNA Purification Kit (produced by Edge Biosystems)).

[0047] PCR was performed using the chromosomal DNA obtained as described above as a template and the primer DNAs (mRecA-F3, mRecA-R3) shown in SEQ ID NOS: 1 and 2. As for the reaction condition, a cycle of denaturation at 94° C. for 10 seconds, annealing at 55° C. for 30 seconds and extension reaction at 70° C. for 2 minutes was repeated for 28 cycles. In addition, a commercially available kit (Pyrobest Taq (produced by Takara Bio Inc.)) was used as a heat-resistant DNA polymerase.

[0048] A DNA fragment of about 1.3 kilobase pairs (“kbp”) containing the recA gene was obtained by PCR as described above. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 19, and the encoded amino acid sequence is shown in SEQ ID NO: 20. Both ends of this DNA fragment were blunt-ended and phosphorylated using BKL Kit (Takara Bio Inc.). The plasmid pUCl9 (Takara Bio Inc.) was treated with the restriction enzyme BamHI and then similarly blunt-ended, and the 5′ phosphate of the digested ends were dephosphorylated.

[0049] The above two DNA fragments were ligated using Ligation Kit (Takara Bio Inc.) to construct pUC-MrecA 1. The direction of the recA gene in this plasmid was the same as the direction of transcription from the lac promoter in the plasmid.

[0050] Then, pUC-MrecAl was digested with the restriction enzyme BamHIl, and the digested ends were further dephosphorylated to prepare a DNA fragment in which the recA gene was split. Furthermore, the plasmid pUC4K (produced by Amersham Biosciences) was treated with the restriction enzyme BamHI to prepare a DNA fragment (1.3 kbp) containing the kanamycin resistance gene (Km^(R)). Both DNA fragments mentioned above were ligated using Ligation Kit to obtain pUC-MrecA1::km.

[0051] The plasmid pUC-MrecA1::km was digested with the restriction enzymes XbaI and KpnI, and the digested product was subjected to electrophoresis to purify and obtain a recA gene DNA fragment inserted with the kanamycin resistance gene (recA::Km^(R)). This DNA fragment was concentrated and desalted by ethanol precipitation.

[0052] The Methylophilus methylotrophus AS1 strain was cultured at 37° C. for 16 hours with shaking in the SEII liquid medium (methanol concentration: 0.5% (v/v)), and 20 mL of the culture broth was centrifuged at 10,000 rpm for 10 minutes to collect the cells. 1 mM HEPES buffer (pH 7.2, 20 ml) was added to the cells to suspend the cells in the buffer, and the suspension was centrifuged. This operation was repeated twice, and 1 ml of the same buffer was finally added to the cells to prepare a cell suspension as electro cells for electroporation.

[0053] About 1 μg of the aforementioned recA gene DNA fragment was added to 100 μL of the electro cells, and an electric pulse was applied to the cells at 18.5 kV/cm, 25 μF and 200 Ω to perform the electroporation. This cell suspension was immediately added to the SEII liquid medium and cultured at 37° C. for 3 hours. Then, this culture broth was applied to a SEII +Km agar medium (SEII medium containing 20 μg/ml of kanamycin and 1.5% (w/v) of agar), and the cells were cultured at 37° C. for three days. As a result, fifty transformants of kanamycin resistant were obtained. Furthermore, seven strains among them were spread again on the SEII +Km agar medium to further purify the colonies. Chromosomal DNA was extracted from one colony, and the structure of the recA gene was analyzed by PCR. The DNA primers were mRecA-F2, mRecA-R2, Km4-F1 and Km4-R1 (having the sequences of SEQ ID NOS: 3, 4, 5 and 6, respectively). First, when PCR was performed using mRecA-F2 and Km4-R1 as a pair of DNA primers and the genomic DNA of the candidate strain as a template, it was confirmed that a DNA fragment having a size of 1530 bp was amplified (the reaction conditions were 94° C. for 10 seconds for denaturation, 50C for 30 seconds for annealing reaction and 72° C. for 2 minutes for extension reaction). Furthermore, when Km4-F1 and mRecA-R2 were used, a DNA fragment having a size of 1950 bp was amplified. This indicated that the recA gene region on the genome of the candidate strain was disrupted by the KM^(R) gene.

[0054] Furthermore, the phenotype of the aforementioned candidate strain with disruption of the recA gene was comfirmed. That is, the recA gene product is an enzyme involved in homologous recombination of DNA and also is involved in the SOS repair mechanism of the cells. Therefore, if the recA gene of the strain was disrupted, and thus the recA function eliminated, the strain would show high sensitivity to ultraviolet irradiation (UV). Therefore, the UV sensitivity of the candidate strain was compared with that of the wild-type strain AS1.

[0055] A candidate for recA-disrupted strain and the AS1 strain were each cultured in 3 mL of the SEII medium for 16 hours, and a part of the culture broth, 300 μL, was inoculated to 3 mL of the same medium. Subsequently, the cells were cultured at 37° C. until the cells reached the logarithmic phase (OD is about 0.5). Then, this culture broth was serially diluted with the SEII liquid medium to prepare dilutions diluted to a degree of 106 times from the original culture broth (1-fold dilution). Furthermore, 15 μL of each dilution was spotted to a surface of the SEII agar medium plate, left for a while to allow each cell suspension to infiltrate into the agar, and dried. Then, the agar plate spotted with the cell suspension was placed under a UV light (Toshiba germicidal lamp, GL15) at a distance of 80 cm and irradiated with a UV ray for 15 seconds. Separately, control plots were also prepared in which UV irradiation was not performed. Both of these agar plates were incubated at 37° C. for one day, and the formation of colonies of each bacterium produced from the spotting site was observed. As a result, it was found that, as expected, the candidate strain of the recA disrupted strain showed UV sensitivity about 1000 times higher than that of the AS1 strain, and thus the obtained kanamycin resistant strain was a recA-disrupted strain also by phenotype.

[0056] Thus, disruption of the recA gene in Methylophilus methylotrophus using a linear DNA was confirmed.

Example 2 Method for Disrupting MtdA Gene (Methylene Tetrahydromethanopterin Tetrahydrofolic Acid Dehydrogenase Gene) Using Linear DNA

[0057] Chromosomal DNA was prepared from the AS1 strain in the same manner as in Example 1. The chromosomal DNA was used as a template, and MmtdA-F 1 and MmtdA-R1 (SEQ ID NOS: 11 and 12, respectively) were used as DNA primers to perform PCR using a commercially available kit, Pyrobest Taq (produced by Takara Bio Inc.) (reaction conditions: denaturation at 94° C. for 10 seconds, annealing at 50° C. for 30 second, and extension reaction at 70° C. for 3 minutes). As a result, a DNA fragment of the mtdA gene having a length of about 2.1 kbp was amplified. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 21, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 22. Subsequently, this DNA fragment was digested with the restriction enzymes BamHI and Sall and then purified.

[0058] Separately, a general-purpose plasmid vector, pBluescriptIlSK-(Stratagene), was similarly digested with BamHI and Sall. This vector fragment and the aforementioned mtda gene fragment were ligated using Ligation Kit to construct pBS-MmtdAl. Then, this plasmid was digested with the restriction enzymes EcoRV and MAul to prepare a DNA fragment in which the mtdA gene region was split.

[0059] Furthermore, the DNA primers for PCR, Km4-F2 and Km4-R2 (shown in SEQ ID NOS: 7 and 8, respectively) were produced and PCR was performed using these primes and pUC4K2 as a template (conditions: denaturation at 94° C. 10 seconds, annealing at 50° C. for 30 seconds, and extension reaction at 70° C. for 1.5 minutes) to amplify a DNA fragment carrying the Km^(R) (kanamycin resistance) gene. Furthermore, the both ends of this DNA fragment were digested with EcoRV and Mlul, and the DNA fragment was purified.

[0060] The two aforementioned fragments were ligated using Ligation Kit to construct pBS-MmtdA1Δ, and the resulting plasmid pBS-MmtdA1 Δ was digested with the restriction enzymes BamHI and SalI to prepare a mtdA::Km^(R) gene fragment consisting of the mtdA gene in which the Km^(R) gene was inserted. This digestion product was concentrated by ethanol precipitation and further subjected to a desalting treatment, and the resultant was used as a DNA sample for electroporation.

[0061] In the same manner as in Example 1, the aforementioned DNA sample was introduced into the AS1 strain by electroporation to obtain about 50 strains of transformants as Km^(R) strains. Six strains were selected from these, and the genomic DNA of each candidate strain was used as a template to perform PCR (conditions: denaturation at 94° C. 10 seconds, annealing at 50° C. for 30 second, and extension reaction at 72° C. for 2.5 minutes) to examine the structure of the mtdA gene region of each candidate strain. The DNA primers used for PCR for this assay were MmtdA-F2, MmtdA-R2, Km4-F1 and Km4-R1 (SEQ ID NOS: 9, 10, 5 and 6, respectively). As a result, a DNA fragment having a size of 2 kbp and a DNA fragment having a size of 1.6 kbp were amplified with the combination of MmtdA-F2 and Km4-R1 and the combination of MmtdA-R2 and Km4-F 1, respectively, as expected, and thus the deficiency of the mtdA gene, which was the target gene of the disruption, was confirmed.

Example 3 Disruption of Mch Gene (Methenyltetrahydromethanopterin Cyclohydrolase Gene) Using Linear DNA

[0062] Chromosomal DNA was prepared from the AS1 strain in the same manner as in Example 1. This DNA was used as a template, and Mmch-F1 and Mmch-R1 (SEQ ID NOS: 13 and 14, respectively) were used as DNA primers to perform PCR (reaction conditions: denaturation at 94° C. for 10 seconds, annealing at 50° C. for 30 second, and extension reaction at 70° C. for 2 minutes). As a result, a DNA fragment of the mch gene having a length of about 1.8 kbp was amplified. The nucleotide sequence of this DNA fragment is shown in SEQ ID NO: 23, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 24. Subsequently, this DNA fragment was digested with the restriction enzymes BamHI and SalI and then purified.

[0063] Separately, a general-purpose plasmid vector, pBluescriptIISK-(Stratagene), was similarly digested with BamHI and SalI. This vector fragment and the aforementioned mch gene fragment were ligated using Ligation Kit to construct pBS-Mmch1. Then, the obtained plasmid was digested with the restriction enzymes EcoRI and PstI to prepare a DNA fragment in which the mch gene region was split.

[0064] Furthermore, the DNA primers for PCR, Km4-F3 and Km4-R3 (shown in SEQ ID NOS: 15 and 16, respectively) were produced and PCR was performed using the primers and pUC4K2 as a template (conditions: denaturation at 94° C. 10 seconds, annealing at 50° C. for 30 seconds, and extension reaction at 70° C. for 1.5 minutes) to amplify a DNA fragment carrying Km^(R) (kanamycin resistance) gene. Furthermore, the both ends of this DNA fragment were digested with EcoRI and PstI, and the DNA fragment was purified.

[0065] The aforementioned two fragments were ligated using Ligation Kit to construct pBS-Mmch1 Δ, and the plasmid pBS-Mmch1 Δ was digested with the restriction enzymes BamHI and SalI to prepare a mch::Km^(R) gene fragment consisting of the mch gene in which the Km^(R) gene was inserted. This digestion product was concentrated by ethanol precipitation and further subjected to a desalting treatment, and the resultant was used as a DNA sample for electroporation.

[0066] In the same manner as in Example 1, the aforementioned DNA sample was introduced into the AS1 strain by electroporation to obtain about 50 strains of transformants as Km^(R) strains. Six strains were selected from them, and the genomic DNA of each candidate strain was used as a template to perform PCR (conditions: denaturation at 94° C. for 10 seconds, annealing at 50° C. for 30 second, and extension reaction at 72° C. for 1.5 minutes) to examine the structure of the mch gene region of each strain. The DNA primers used for PCR were Mmch-F2, Mmch-R2, Km4-F1 and Km4-R1 (SEQ ID NOS: 17, 18, 5 and 6, respectively). As a result, amplification of a DNA fragment having a size of 1.8 kbp and a DNA fragment having a size of 2.5 kbp was confirmed with the combination of Mmch-F2 and Km4-R1 and the combination of Mmch-R2 and Km4-F1, respectively, as expected, and thus it was confirmed that strains deficient in mch, which was the target gene of the disruption, were prepared.

[0067] While the invention has been described in detail with reference to preferred embodiments thereof, it will be apparent to one skilled in the art that various changes can be made, and equivalents employed, without departing from the scope of the invention. Each of the aforementioned documents, including the foreign priority document JP2003-1927, is incorporated by reference herein in its entirety.

1 24 1 18 DNA Artificial sequence primer 1 ggaaaacata ataatgct 18 2 24 DNA Artificial sequence primer 2 ttcgcgcttg cttagatact ccag 24 3 30 DNA Artificial sequence primer 3 gaccagttat ctccggcttg tgcattgaaa 30 4 27 DNA Artificial sequence primer 4 tcgtccgcat aacccttgag cttttgc 27 5 33 DNA Artificial sequence primer 5 agactaaact ggctgacgga atttatgcct ctt 33 6 35 DNA Artificial sequence primer 6 ttggtgattt tgaacttttg ctttgccacg gaacg 35 7 45 DNA Artificial sequence primer 7 cttgatatcg ctagctcgta tgttgtgtgg aattgtgagc ggata 45 8 39 DNA Artificial sequence primer 8 accaacgcgt aatcgcccca tcatccagcc agaaagtga 39 9 24 DNA Artificial sequence primer 9 tgggtttgtg gtagatagtg ggcg 24 10 24 DNA Artificial sequence primer 10 gcgcttttat caatggcaac cctg 24 11 34 DNA Artificial sequence primer 11 gccaggatcc ctgaccgcca cacaagttat ccag 34 12 35 DNA Artificial sequence primer 12 acctgtcgac gatgcaactc gccctcatgc cagat 35 13 35 DNA Artificial sequence primer 13 ggcaggatcc ttgattggcg tacatgcact caagc 35 14 34 DNA Artificial sequence primer 14 atatgtcgac gcgtgatgat ttgctgggtg gtgc 34 15 27 DNA Artificial sequence primer 15 acagaattcc aaggggtgtt atgagcc 27 16 24 DNA Artificial sequence primer 16 gtgtcggggc tggcttaact atgc 24 17 27 DNA Artificial sequence primer 17 gggtcaaaaa tccgcaatgg ctgaaaa 27 18 27 DNA Artificial sequence primer 18 agcacgtcag caatctcaaa tggggtg 27 19 1315 DNA Methylophilus methylotrophus CDS (219)..(1247) 19 ggaaaacata ataatgctcc cagattccct taacgtgtca tcctgctcaa ggttgacacg 60 attcaggccg aatttttaag ccgaaatcac tggcttgaat tcatggcttc gccatttcac 120 tgatggtcga tttatgaaat aattagaagt tatggtttgg attatatagc gtttgtataa 180 acagcgcatt tttatcgcta ataaagaagg taatcagc atg gat gac aac aaa agc 236 Met Asp Asp Asn Lys Ser 1 5 aaa gcg ctc gcc gcc gca ctt tcc caa att gaa aaa cag ttc ggt aaa 284 Lys Ala Leu Ala Ala Ala Leu Ser Gln Ile Glu Lys Gln Phe Gly Lys 10 15 20 ggc tcc att atg cgc atg ggc gat gct gat atc ggc gaa gac ctg caa 332 Gly Ser Ile Met Arg Met Gly Asp Ala Asp Ile Gly Glu Asp Leu Gln 25 30 35 gtg gtt tcc acc ggc tca ctg ggc ctg gat atc gca ctg ggg gtg ggt 380 Val Val Ser Thr Gly Ser Leu Gly Leu Asp Ile Ala Leu Gly Val Gly 40 45 50 ggc ttg cca cgt ggc cgt att atc gaa att tat ggc cct gag tct tcc 428 Gly Leu Pro Arg Gly Arg Ile Ile Glu Ile Tyr Gly Pro Glu Ser Ser 55 60 65 70 ggt aaa acc aca ttg acc ttg tcc gcg att gcc gaa atg caa aag ttg 476 Gly Lys Thr Thr Leu Thr Leu Ser Ala Ile Ala Glu Met Gln Lys Leu 75 80 85 ggc ggt gtc gca gca ttt atc gat gct gag cat gct ctg gat cca cag 524 Gly Gly Val Ala Ala Phe Ile Asp Ala Glu His Ala Leu Asp Pro Gln 90 95 100 tac gcg gcc aag ctg ggc gtg aat gtg cct gaa tta ctg att tca cag 572 Tyr Ala Ala Lys Leu Gly Val Asn Val Pro Glu Leu Leu Ile Ser Gln 105 110 115 cct gac acc ggg gag caa gcg ttg gaa att gcc gat atg ctg gta cgc 620 Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile Ala Asp Met Leu Val Arg 120 125 130 tcc ggc tcc gtg gat atc gtg gtt gtt gac tcg gtg gct gcc ttg acc 668 Ser Gly Ser Val Asp Ile Val Val Val Asp Ser Val Ala Ala Leu Thr 135 140 145 150 cca cgt gcc gaa att gaa ggt gaa atg ggt gac agc cac atg ggc ttg 716 Pro Arg Ala Glu Ile Glu Gly Glu Met Gly Asp Ser His Met Gly Leu 155 160 165 cag gca cgc ctg atg tca cag gca ttg cgt aag ctc act ggt aac atc 764 Gln Ala Arg Leu Met Ser Gln Ala Leu Arg Lys Leu Thr Gly Asn Ile 170 175 180 aag cgt acc aat acg ctg gtg att ttt atc aac cag atc cgt atg aag 812 Lys Arg Thr Asn Thr Leu Val Ile Phe Ile Asn Gln Ile Arg Met Lys 185 190 195 atc ggt gtc atg ttc ggt aat cct gaa acg acc act ggc ggt aac gcg 860 Ile Gly Val Met Phe Gly Asn Pro Glu Thr Thr Thr Gly Gly Asn Ala 200 205 210 ctc aag ttt tac tct tct gtc cgt ctc gat atc cgc cgt acc ggt gcg 908 Leu Lys Phe Tyr Ser Ser Val Arg Leu Asp Ile Arg Arg Thr Gly Ala 215 220 225 230 att aaa aaa ggc gac gag gtg att ggc tct gag acc aag gtg aag gtc 956 Ile Lys Lys Gly Asp Glu Val Ile Gly Ser Glu Thr Lys Val Lys Val 235 240 245 atc aag aac aag gtt gcg ccg ccg ttc aag cag gct gaa ttc gac atc 1004 Ile Lys Asn Lys Val Ala Pro Pro Phe Lys Gln Ala Glu Phe Asp Ile 250 255 260 atg tac ggc gaa ggt att tcc cgt ctg ggc gaa atc att gag ttg ggt 1052 Met Tyr Gly Glu Gly Ile Ser Arg Leu Gly Glu Ile Ile Glu Leu Gly 265 270 275 aca aat ttg aaa ctg gtt gag aaa tca ggt gcg tgg tac agc tac aac 1100 Thr Asn Leu Lys Leu Val Glu Lys Ser Gly Ala Trp Tyr Ser Tyr Asn 280 285 290 ggt gaa aaa atc ggc cag ggt aaa gaa aac gct aaa gag ttc ctg cgc 1148 Gly Glu Lys Ile Gly Gln Gly Lys Glu Asn Ala Lys Glu Phe Leu Arg 295 300 305 310 gag aat cca gcg att gcg gca gaa att gaa gcc aag att cgc gac aac 1196 Glu Asn Pro Ala Ile Ala Ala Glu Ile Glu Ala Lys Ile Arg Asp Asn 315 320 325 tct aat gtg ctg gca gat agc atg act gcg gcc aga agt gag gac gat 1244 Ser Asn Val Leu Ala Asp Ser Met Thr Ala Ala Arg Ser Glu Asp Asp 330 335 340 taa gcagcttgcg tcagccgatt gaaaagtcgc tgcgccagcg tgcgctggag 1297 tatctaagca agcgcgaa 1315 20 342 PRT Methylophilus methylotrophus 20 Met Asp Asp Asn Lys Ser Lys Ala Leu Ala Ala Ala Leu Ser Gln Ile 1 5 10 15 Glu Lys Gln Phe Gly Lys Gly Ser Ile Met Arg Met Gly Asp Ala Asp 20 25 30 Ile Gly Glu Asp Leu Gln Val Val Ser Thr Gly Ser Leu Gly Leu Asp 35 40 45 Ile Ala Leu Gly Val Gly Gly Leu Pro Arg Gly Arg Ile Ile Glu Ile 50 55 60 Tyr Gly Pro Glu Ser Ser Gly Lys Thr Thr Leu Thr Leu Ser Ala Ile 65 70 75 80 Ala Glu Met Gln Lys Leu Gly Gly Val Ala Ala Phe Ile Asp Ala Glu 85 90 95 His Ala Leu Asp Pro Gln Tyr Ala Ala Lys Leu Gly Val Asn Val Pro 100 105 110 Glu Leu Leu Ile Ser Gln Pro Asp Thr Gly Glu Gln Ala Leu Glu Ile 115 120 125 Ala Asp Met Leu Val Arg Ser Gly Ser Val Asp Ile Val Val Val Asp 130 135 140 Ser Val Ala Ala Leu Thr Pro Arg Ala Glu Ile Glu Gly Glu Met Gly 145 150 155 160 Asp Ser His Met Gly Leu Gln Ala Arg Leu Met Ser Gln Ala Leu Arg 165 170 175 Lys Leu Thr Gly Asn Ile Lys Arg Thr Asn Thr Leu Val Ile Phe Ile 180 185 190 Asn Gln Ile Arg Met Lys Ile Gly Val Met Phe Gly Asn Pro Glu Thr 195 200 205 Thr Thr Gly Gly Asn Ala Leu Lys Phe Tyr Ser Ser Val Arg Leu Asp 210 215 220 Ile Arg Arg Thr Gly Ala Ile Lys Lys Gly Asp Glu Val Ile Gly Ser 225 230 235 240 Glu Thr Lys Val Lys Val Ile Lys Asn Lys Val Ala Pro Pro Phe Lys 245 250 255 Gln Ala Glu Phe Asp Ile Met Tyr Gly Glu Gly Ile Ser Arg Leu Gly 260 265 270 Glu Ile Ile Glu Leu Gly Thr Asn Leu Lys Leu Val Glu Lys Ser Gly 275 280 285 Ala Trp Tyr Ser Tyr Asn Gly Glu Lys Ile Gly Gln Gly Lys Glu Asn 290 295 300 Ala Lys Glu Phe Leu Arg Glu Asn Pro Ala Ile Ala Ala Glu Ile Glu 305 310 315 320 Ala Lys Ile Arg Asp Asn Ser Asn Val Leu Ala Asp Ser Met Thr Ala 325 330 335 Ala Arg Ser Glu Asp Asp 340 21 2158 DNA Methylophilus methylotrophus CDS (514)..(1407) 21 gccaggatcc ctgaccgcca cacaagttat ccagcaacag ctgttaagcc agggtttgcc 60 tgccttgatc gagcaggatt ttgccacgtt cagccgtttt cttggtgatt tgcaagctta 120 taatgcagat tatttcgcgc ctgcacaggg tggtgcctat gccagttcga gcgtggcaag 180 cattttgcaa tctattaaaa aacaaggtta tgcaggcatc ggacagacgt cctggggacc 240 aacaggattt gtgttgctgc cttcgcgtgc agaagcggtc actatgcaaa tgcagctgct 300 gcatttgcat gctaacgatg cctccctggg atttatcgtc acagcagcca tgaatcagtc 360 ggccaatatt atgtttggga atggcgcaga ttaaattttc ttaagataat tttgaaaagt 420 tatggctttt acggtctact ctttattttg aactggtctg gatatgtgta tattggcgaa 480 agatattgtt aacgaaccgt accgggggga aga atg aaa aaa acc agt att atg 534 Met Lys Lys Thr Ser Ile Met 1 5 cat ttg ttc act gct gcc aag aat gcc agt cca ttt gat gtg aat atg 582 His Leu Phe Thr Ala Ala Lys Asn Ala Ser Pro Phe Asp Val Asn Met 10 15 20 gcc ttt gat gct ggc tat gag aaa att att tct tac acc gat gtg act 630 Ala Phe Asp Ala Gly Tyr Glu Lys Ile Ile Ser Tyr Thr Asp Val Thr 25 30 35 ttg aat gaa atc gtc gcg ttg acg cag gat gcc att ttt tca cgc agc 678 Leu Asn Glu Ile Val Ala Leu Thr Gln Asp Ala Ile Phe Ser Arg Ser 40 45 50 55 ccg agt gga tta aag cag caa gcc tta ttt ttt ggt ggc cgc gat atc 726 Pro Ser Gly Leu Lys Gln Gln Ala Leu Phe Phe Gly Gly Arg Asp Ile 60 65 70 cag gtg gcg ctg gaa atg cag aag cag gcg cgc agt gcc atg ttc aag 774 Gln Val Ala Leu Glu Met Gln Lys Gln Ala Arg Ser Ala Met Phe Lys 75 80 85 cca ttt gaa tgc cat act ttt tct gat ccg tcc ggt gcc ttt acc acg 822 Pro Phe Glu Cys His Thr Phe Ser Asp Pro Ser Gly Ala Phe Thr Thr 90 95 100 gca gca gcc atg ctg gcc aaa gtc gat ttt tat ttg cag aaa tct ggt 870 Ala Ala Ala Met Leu Ala Lys Val Asp Phe Tyr Leu Gln Lys Ser Gly 105 110 115 agt ggt ttg ggc aag gaa aaa gtc gct att ttt ggt gcc agt ggt acc 918 Ser Gly Leu Gly Lys Glu Lys Val Ala Ile Phe Gly Ala Ser Gly Thr 120 125 130 135 gtg ggc tcg aca gca gca ctc atc gca gct cgc cag gga gcc act gta 966 Val Gly Ser Thr Ala Ala Leu Ile Ala Ala Arg Gln Gly Ala Thr Val 140 145 150 ttg atg gtg gcg cac tcg gat gtt gcc agt atg cag gcg tat gtt gat 1014 Leu Met Val Ala His Ser Asp Val Ala Ser Met Gln Ala Tyr Val Asp 155 160 165 aag ctt tct agc aat tat gat gtc agc ctc aaa gta gtg gat ggc agt 1062 Lys Leu Ser Ser Asn Tyr Asp Val Ser Leu Lys Val Val Asp Gly Ser 170 175 180 aca gag gct gcc aaa gtg gct gtg ttg aat gaa gcg aca gta gcc ttg 1110 Thr Glu Ala Ala Lys Val Ala Val Leu Asn Glu Ala Thr Val Ala Leu 185 190 195 tgt gca aca cca gct ggg att cgc gtc ctt gaa atc aag caa ttc gcc 1158 Cys Ala Thr Pro Ala Gly Ile Arg Val Leu Glu Ile Lys Gln Phe Ala 200 205 210 215 aac tcc aaa tca ctg aaa gtg gtg gca gac gta aac gca gtc cct cct 1206 Asn Ser Lys Ser Leu Lys Val Val Ala Asp Val Asn Ala Val Pro Pro 220 225 230 tct ggc att gag ggc gta gac aca ttc tct gat ggt ggc gtg att gaa 1254 Ser Gly Ile Glu Gly Val Asp Thr Phe Ser Asp Gly Gly Val Ile Glu 235 240 245 ggc aca caa gtg gcc ggt ttt ggc gcc ttg gcg att ggc cag ttg aaa 1302 Gly Thr Gln Val Ala Gly Phe Gly Ala Leu Ala Ile Gly Gln Leu Lys 250 255 260 tat gtc acc caa aac aag cta ctg gag caa atg ctg caa agc gaa agc 1350 Tyr Val Thr Gln Asn Lys Leu Leu Glu Gln Met Leu Gln Ser Glu Ser 265 270 275 ccc atg cac att gat tac cat gag gca tat gag tat gcc tgt gca cac 1398 Pro Met His Ile Asp Tyr His Glu Ala Tyr Glu Tyr Ala Cys Ala His 280 285 290 295 gtg gag taa agcgattctt gcgattggct gtattgtcac agagtgcgcg 1447 Val Glu tatttatagc cagatggcgc aacaagaagg ctttagtgta ttggctgtgg acgcgtttgc 1507 ggataacgat acgcagcaat ctgcaacatt ggtataccac tggccaggcc tgtgcggacc 1567 ggatgtcaac aatgaaatgt ctgggttaat ggaagtattg gatagtttca agccggatgc 1627 cgttttactt ggttctggtt ttgaagcaga tcaagcggca tatgcaaagt tattcacacg 1687 ccatgcaata tttggcaata caccggaaac cgtggcccgg gtcaaaaatc cgcaatggct 1747 gaaaaattat tgtgatgcgc acggcgtcca gtcgccatgc atcgccacgc aaaagccggt 1807 cgaaggtcgt tggctgcata aacaggcggg acgatgtggt ggtatgcatg tgcaagactg 1867 gtcacctgca gcaacagtca ctgcaaaaag ttactggcaa gcatttcagc caggacaagc 1927 cgtgggaata ttgtttgtcg cgcatcagca ggcattcaca ttgattggcg tacatgcact 1987 caagcaacgc gcagggagct atgcttatgc aggcgtgaag cgcttgcatg atccagcgct 2047 aactgtcgct gccacagagt tattgcaggc agtcttgcca ggcttgggat tagttggcat 2107 taacagtatt gatgccatct ggcatgaggg cgagttgcat cgtcgacagg t 2158 22 297 PRT Methylophilus methylotrophus 22 Met Lys Lys Thr Ser Ile Met His Leu Phe Thr Ala Ala Lys Asn Ala 1 5 10 15 Ser Pro Phe Asp Val Asn Met Ala Phe Asp Ala Gly Tyr Glu Lys Ile 20 25 30 Ile Ser Tyr Thr Asp Val Thr Leu Asn Glu Ile Val Ala Leu Thr Gln 35 40 45 Asp Ala Ile Phe Ser Arg Ser Pro Ser Gly Leu Lys Gln Gln Ala Leu 50 55 60 Phe Phe Gly Gly Arg Asp Ile Gln Val Ala Leu Glu Met Gln Lys Gln 65 70 75 80 Ala Arg Ser Ala Met Phe Lys Pro Phe Glu Cys His Thr Phe Ser Asp 85 90 95 Pro Ser Gly Ala Phe Thr Thr Ala Ala Ala Met Leu Ala Lys Val Asp 100 105 110 Phe Tyr Leu Gln Lys Ser Gly Ser Gly Leu Gly Lys Glu Lys Val Ala 115 120 125 Ile Phe Gly Ala Ser Gly Thr Val Gly Ser Thr Ala Ala Leu Ile Ala 130 135 140 Ala Arg Gln Gly Ala Thr Val Leu Met Val Ala His Ser Asp Val Ala 145 150 155 160 Ser Met Gln Ala Tyr Val Asp Lys Leu Ser Ser Asn Tyr Asp Val Ser 165 170 175 Leu Lys Val Val Asp Gly Ser Thr Glu Ala Ala Lys Val Ala Val Leu 180 185 190 Asn Glu Ala Thr Val Ala Leu Cys Ala Thr Pro Ala Gly Ile Arg Val 195 200 205 Leu Glu Ile Lys Gln Phe Ala Asn Ser Lys Ser Leu Lys Val Val Ala 210 215 220 Asp Val Asn Ala Val Pro Pro Ser Gly Ile Glu Gly Val Asp Thr Phe 225 230 235 240 Ser Asp Gly Gly Val Ile Glu Gly Thr Gln Val Ala Gly Phe Gly Ala 245 250 255 Leu Ala Ile Gly Gln Leu Lys Tyr Val Thr Gln Asn Lys Leu Leu Glu 260 265 270 Gln Met Leu Gln Ser Glu Ser Pro Met His Ile Asp Tyr His Glu Ala 275 280 285 Tyr Glu Tyr Ala Cys Ala His Val Glu 290 295 23 1823 DNA Methylophilus methylotrophus CDS (522)..(1496) 23 ggcaggatcc ttgattggcg tacatgcact caagcaacgc gcagggagct atgcttatgc 60 aggcgtgaag cgcttgcatg atccagcgct aactgtcgct gccacagagt tattgcaggc 120 agtcttgcca ggcttgggat tagttggcat taacagtatt gatgccatct ggcatgaggg 180 cgagttgcat ctcatcgagg tgaacccccg actcagcgcc agtatgcgtc tgtatgcagg 240 gttgccattg ataaaagcgc atatggacag ttgcaatggc aacatcatgc ctctgcaaca 300 acatactaaa acgcatgcct gccattgcat tgcgtatgca cgacaagaga ttaacgcaag 360 tcatctagac tttcctgact ggttggaaga ccggcccagt ggtggcatga ttgctgcggg 420 tctgcccgtt tgcagtctat atgcacaagg ggactcagac agggaattgc tacaggcttt 480 gcaagataag aaaacacgat tagagaaact atgggggact t atg tct gta acc gca 536 Met Ser Val Thr Ala 1 5 tcg aat tca aca tcc att agc gtt caa caa tat agc gca cca ctg gtg 584 Ser Asn Ser Thr Ser Ile Ser Val Gln Gln Tyr Ser Ala Pro Leu Val 10 15 20 gcg cat ctg atg gcc aat gcc cca gct tta ggc tgc gca gtg gca acg 632 Ala His Leu Met Ala Asn Ala Pro Ala Leu Gly Cys Ala Val Ala Thr 25 30 35 cat gaa aca ggc gcc acg att gtg gat gca ggt att caa gca act ggc 680 His Glu Thr Gly Ala Thr Ile Val Asp Ala Gly Ile Gln Ala Thr Gly 40 45 50 ggc ctg gaa gca ggg cgc atc atc gcc gaa att tgc atg ggt ggt tta 728 Gly Leu Glu Ala Gly Arg Ile Ile Ala Glu Ile Cys Met Gly Gly Leu 55 60 65 ggt aga gtg tcg ttg cag caa gtg ccg caa ttt gcc cac tgg cct ctc 776 Gly Arg Val Ser Leu Gln Gln Val Pro Gln Phe Ala His Trp Pro Leu 70 75 80 85 agt gtc gtg gtg aca gct acc caa ccg gtg att gcc tgc ctt ggc agt 824 Ser Val Val Val Thr Ala Thr Gln Pro Val Ile Ala Cys Leu Gly Ser 90 95 100 cag tat gcc ggc tgg gcc ttg tca cac gaa aaa ttc ttc tca ctg ggc 872 Gln Tyr Ala Gly Trp Ala Leu Ser His Glu Lys Phe Phe Ser Leu Gly 105 110 115 agt ggc ccg gca cgc tca att gca cag cgt gaa gaa gtc ttc aaa gat 920 Ser Gly Pro Ala Arg Ser Ile Ala Gln Arg Glu Glu Val Phe Lys Asp 120 125 130 att aat tac agt gat aaa ggc gag caa acg gtt ttg gtg ctg gaa acc 968 Ile Asn Tyr Ser Asp Lys Gly Glu Gln Thr Val Leu Val Leu Glu Thr 135 140 145 gac aag gtg cct cct gtg cag gtg att gaa aaa gtg gcc aga gat act 1016 Asp Lys Val Pro Pro Val Gln Val Ile Glu Lys Val Ala Arg Asp Thr 150 155 160 165 ggc ctg cca gcc aat aag ctg aca ttt atc ctg acc cca acc cgc agt 1064 Gly Leu Pro Ala Asn Lys Leu Thr Phe Ile Leu Thr Pro Thr Arg Ser 170 175 180 gtg gcc ggt tcc ttg caa gtg act gca cgt gtg ctc gaa gtt gca ctg 1112 Val Ala Gly Ser Leu Gln Val Thr Ala Arg Val Leu Glu Val Ala Leu 185 190 195 cat aaa tgc cat gcc ttg cat ttt gac ctg aat gcc att gtc gat ggt 1160 His Lys Cys His Ala Leu His Phe Asp Leu Asn Ala Ile Val Asp Gly 200 205 210 tat ggt gtc gcg cca gta ccg gcg ccc tcg cca gac ttt atc gtc ggc 1208 Tyr Gly Val Ala Pro Val Pro Ala Pro Ser Pro Asp Phe Ile Val Gly 215 220 225 atg ggc cgt acc aat gat gcg atc ctg ttt ggc ggc ttt gtg cag ttg 1256 Met Gly Arg Thr Asn Asp Ala Ile Leu Phe Gly Gly Phe Val Gln Leu 230 235 240 245 ttt gtg aat acc gat gat gct gca gcg gaa caa ctc gcc cag caa cta 1304 Phe Val Asn Thr Asp Asp Ala Ala Ala Glu Gln Leu Ala Gln Gln Leu 250 255 260 cct tcc tct tca tcc aaa gat tac ggc cgc cca ttc gca cag gtg ttc 1352 Pro Ser Ser Ser Ser Lys Asp Tyr Gly Arg Pro Phe Ala Gln Val Phe 265 270 275 aaa gcc gtt aat atg gac ttt tac cag att gac ccc atg ttg ttc tct 1400 Lys Ala Val Asn Met Asp Phe Tyr Gln Ile Asp Pro Met Leu Phe Ser 280 285 290 cca gcc aaa gtc agt gtg act aac ctc aag tcc ggc aag act ttc ttt 1448 Pro Ala Lys Val Ser Val Thr Asn Leu Lys Ser Gly Lys Thr Phe Phe 295 300 305 ggc ggc cag ttt aat gaa acc ctt ctg aat caa tca ttt gga agt taa 1496 Gly Gly Gln Phe Asn Glu Thr Leu Leu Asn Gln Ser Phe Gly Ser 310 315 320 atttaaggtg ctataaaagt tcttgacgcg ggattctgtg caaaatgcat gggtcccgcg 1556 tgatcatttc aacgctgaca tgaacgtcct tcctattttt accgacgaag tagcccaggc 1616 tggtggctgg cacggacaga gtctggcgca agcctttgca aaacttggct ggcaggcatt 1676 gatggtgtca ttggatagtt gccacgtcag tattgtgaat cagcaggtgc aagtccacat 1736 cccagggctc acacaagctg cacctttggc attcgtgcgt ggcgtggcgg cgggcaccac 1796 ccagcaaatc atcacgcgtc gacatat 1823 24 324 PRT Methylophilus methylotrophus 24 Met Ser Val Thr Ala Ser Asn Ser Thr Ser Ile Ser Val Gln Gln Tyr 1 5 10 15 Ser Ala Pro Leu Val Ala His Leu Met Ala Asn Ala Pro Ala Leu Gly 20 25 30 Cys Ala Val Ala Thr His Glu Thr Gly Ala Thr Ile Val Asp Ala Gly 35 40 45 Ile Gln Ala Thr Gly Gly Leu Glu Ala Gly Arg Ile Ile Ala Glu Ile 50 55 60 Cys Met Gly Gly Leu Gly Arg Val Ser Leu Gln Gln Val Pro Gln Phe 65 70 75 80 Ala His Trp Pro Leu Ser Val Val Val Thr Ala Thr Gln Pro Val Ile 85 90 95 Ala Cys Leu Gly Ser Gln Tyr Ala Gly Trp Ala Leu Ser His Glu Lys 100 105 110 Phe Phe Ser Leu Gly Ser Gly Pro Ala Arg Ser Ile Ala Gln Arg Glu 115 120 125 Glu Val Phe Lys Asp Ile Asn Tyr Ser Asp Lys Gly Glu Gln Thr Val 130 135 140 Leu Val Leu Glu Thr Asp Lys Val Pro Pro Val Gln Val Ile Glu Lys 145 150 155 160 Val Ala Arg Asp Thr Gly Leu Pro Ala Asn Lys Leu Thr Phe Ile Leu 165 170 175 Thr Pro Thr Arg Ser Val Ala Gly Ser Leu Gln Val Thr Ala Arg Val 180 185 190 Leu Glu Val Ala Leu His Lys Cys His Ala Leu His Phe Asp Leu Asn 195 200 205 Ala Ile Val Asp Gly Tyr Gly Val Ala Pro Val Pro Ala Pro Ser Pro 210 215 220 Asp Phe Ile Val Gly Met Gly Arg Thr Asn Asp Ala Ile Leu Phe Gly 225 230 235 240 Gly Phe Val Gln Leu Phe Val Asn Thr Asp Asp Ala Ala Ala Glu Gln 245 250 255 Leu Ala Gln Gln Leu Pro Ser Ser Ser Ser Lys Asp Tyr Gly Arg Pro 260 265 270 Phe Ala Gln Val Phe Lys Ala Val Asn Met Asp Phe Tyr Gln Ile Asp 275 280 285 Pro Met Leu Phe Ser Pro Ala Lys Val Ser Val Thr Asn Leu Lys Ser 290 295 300 Gly Lys Thr Phe Phe Gly Gly Gln Phe Asn Glu Thr Leu Leu Asn Gln 305 310 315 320 Ser Phe Gly Ser 

We claim:
 1. A method for producing a recombinant of a methanol-assimilating bacterium in which an exogenous linear DNA fragment is introduced into the chromosomal DNA of the methanol-assimilating bacterium comprising: (a) preparing an exogenous linear DNA fragment comprising a nucleotide sequence identical to a nucleotide sequence of an arbitrary region of said chromosomal DNA, (b) introducing said linear DNA fragment into the methanol-assimilating bacterium to obtain recombinants, and (c) selecting a recombinant in which said region on the chromosome is replaced with said linear DNA fragment.
 2. The method according to claim 1, wherein said methanol-assimilating bacterium is a Methylophilus bacterium.
 3. The method according to claim 1, wherein said methanol-assimilating bacterium is Methylophilus methylotrophus.
 4. The method according to claim 1, wherein said linear DNA fragment comprises a segment having said nucleotide sequence identical to the arbitrary region of said chromosomal DNA, and another sequence inserted into the segment.
 5. The method according to claim 1, wherein said linear DNA fragment comprises partial deletion or substitution of one or more nucleotides. 